This proposal is intended to elucidate the molecular mechanisms involved in the acute regulation of metabolism by insulin. A complex carbohydrate-phosphate substance has been identified which is generated by the insulin-sensitive phosphodiesteratic hydrolysis of a novel phosphatidylinositol containing glycolipid, and acutely regulates the activities of several insulin sensitive enzymes. This substance has been tentatively identified as an inositol-glycan and thus far fulfills criteria for a second messenger of insulin action. The precise chemical identity of this enzyme-modulating activity will be pursued. Purification procedures will be scaled up to obtain sufficient material for chemical analysis. This will begin by determination of the carbohydrate constituents using distinct hydrolytic techniques with different specificities for particular glycosidic linkages. Component monosaccharides will analyzed by mass spectroscopy. The structure/function relationship of the substance will be evaluated, and its chemical identity will be verified by organic synthesis. The mechanism of generation of the substance will be evaluated by elucidating the biosynthesis of the glycolipid precursor. The hydrolysis of this glycolipid will be evaluated by the purification and characterization of the phospholipase enzyme responsible for the generation of the inositol-glycan. The purified glycolipid precursor and enzyme will be reconstituted into liposomes with the purified insulin receptor to study hormonal regulation of this process. Another product of this insulin-stimulated hydrolysis reaction is diacylglycerol. Its role will be evaluated with respect to insulin action. The metabolism and processing of the carbohydrate enzyme modulator will be studied, and its biochemical actions in the control of protein phosphorylation will be evaluated. It is anticipated that elucidation of the chemical identity and the process by which this second messenger is generated, processed and its effects exerted will contribute to understanding the hormonal regulation of cellular metabolism.